Membrane shape is important not only as a static aspect of size and structure of cells and organelles, but dynamically changes in numerous processes such as membrane signaling and trafficking. At the plasma membrane, the formation of in- and exvaginations, in processes such as endocytosis and the generation of filopodia, respectively, are some of the most important phenomena where membrane curvature is modulated. The discovery of a class of proteins which contain crescent shaped scaffolds called BAR (Bin/amphiphysin/Rvs) domain proteins, has prompted a growing interest in understanding how proteins couple with membrane curvature. BAR domains are found in numerous proteins implicated in human disease, and many contain disease driving mutations and/or show altered expression levels under pathological conditions. Additional peripheral proteins that are related to membrane curvature include intrinsically disordered proteins such as ?-synuclein, as well as ENTH domain-containing proteins such as epsin, both of which are believed to be involved in membrane trafficking phenomena. Endocytosis is the primary mechanism by which pathogens enter cells. To improve the understanding of the mechanism and regulation of this process therefore is a matter of primary biomedical relevance. However, despite the fact that more than 90000 research contributions have investigated endocytosis alone, the mechanisms for initiation of this process are not understood. This is due in part to the fact that in cells numerous endocytic mechanisms operate in parallel and that the degree for experimental control of key parameters in cells is limited. The goal of this project is to understand how membrane shape transitions are regulated in processes such as endocytosis. In order to achieve this goal, we have developed an experimental biophysical model membrane approach that allows us to determine the conditions under which membranes undergo shape transitions. With the help of this tool, which consists of a combined micro- manipulation/fluorescence approach that is presently used exclusively in our laboratory, we will investigate mechanisms of the function of the many proteins involved in endocytosis, and isolate key modulators of membrane shape transitions. We already have developed a theoretical framework that will facilitate mechanistic interpretation of our findings. While plasma membranes experience significant asymmetry with respect to transmembrane ion and lipid distributions, model membrane research has largely focused on symmetric membranes. We will overcome this limitation and determine to what extent membrane asymmetry, which will include cytoskeletal interactions, contributes to the function of peripheral proteins in shaping membranes. Overall this project will provide far-reaching insight into the mechanisms by which peripheral proteins deform membranes under healthy and pathological conditions.